Proper motor circuit formation is necessary for all behavior. However, motor circuit development is poorly understood. My lab is taking a first principles approach to this problem. We are building a bridge between neuron birth from stem cells to mature motor circuit architecture. The Drosophila larval nerve cord is an ideal and uniquely-suited system for these studies because neuronal stem cell lineages and neuronal circuits are map at single cell resolution. Furthermore, Drosophila has been used to generate many fundamental discoveries relevant to neurogenesis in vertebrates, and recent work from my lab revealed remarkable parallels between motor system development in flies and vertebrates. This demonstrates that comparison between different species is essential for identifying fundamental principles underlying motor circuit development. In Aim 1, we discriminate among attractive models of lineage-based motor circuit assembly. The two objectives of this aim are to comprehensively determine lineage, birth time, and synaptic wiring of neurons from a single lineage, NB3-3, as well as the synaptic partners of NB3-3 progeny, and to generate tools to study assembly of circuits containing NB3-3 progeny in real time and in vivo. In Aim 2, we test the hypothesis that Temporal Identity transcription factors (TFs) direct circuit assembly. Temporal Identity TFs cause stem cells to generate different neurons over time. First, we describe new preliminary data in which we manipulate temporal identity TF expression in a single lineage to produce a series of motor neurons with early-born molecular identities at abnormally late times during development. Motor neurons born at the ?wrong? time still form synaptic partnerships with muscles that are characteristic of their molecular identity. These data provide support for the hypothesis that lineage-intrinsic gene expression can control circuit assembly. However, muscles are a stable physical substrate in comparison to neurons in the CNS during neurogenesis. The objective of this aim is to examine the same manipulation in the context of neuron-neuron synapses in the CNS. We use an innovative combination of sophisticated lineage tracing, neuronal stem cell specific gene manipulation, calcium imaging, and electron microscope connectomic approaches. This includes use of cutting-edge ?comparative connectomics?, in which circuit wiring in two different genetic backgrounds can be compared. These innovations allow us to study circuit assembly at single neuron resolution. Achieving single neuron resolution is a significant advance, which is important because this is the level at which wiring decisions are made. The experiments described here are significant because they will reveal fundamental principles of lineage-based motor circuit development. This has wide ranging implications for the genetic basis of behavior, neuro-developmental motor disease, and stem cell therapy for spinal cord injury. Thus, this grant proposal is relevant to the NIH mission.